The present invention relates to Drosophila insulin-like genes and methods for identifying insulin-like genes. The invention provides nucleotide sequences of Drosophila insulin-like genes, amino acid sequences of their encoded proteins (including peptide or polypeptide), and derivatives (e.g., fragments) and analogs thereof. The invention further relates to fragments (and derivatives and analogs thereof) of insulin-like proteins which comprise one or more domains of an insulin-like protein. Antibodies to an insulin-like protein, and derivatives and analogs thereof, are provided. Methods of production of an insulin-like protein (e.g., by recombinant means), and derivatives and analogs thereof, are provided. Methods to identify the biological function of a Drosophila insulin-like gene are provided, including various methods for the functional modification (e.g., overexpression, underexpression, mutation, knock-out) of one gene, or of two or more genes simultaneously. Methods to identify a Drosophila gene which modifies the function of, and/or functions in a downstream pathway from, an insulin-like gene are provided. The invention further provides for use of Drosophila insulin-like proteins as a media additive or pesticide.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
2.1. THE INSULIN SUPERFAMILY
Insulin-like proteins are a large and widely-distributed group of structurally-related peptide hormones that have pivotal roles in controlling animal growth, development, reproduction, and metabolism (Blundell and Humbel, 1980, Nature 287:781-787). Consequently, the insulin superfamily has become one of the most intensively investigated classes of peptide hormones. Such hormones have a vast array of uses including, for example, clinical applications in human disease, management of fish and livestock, and the control of agriculturally-important animal pests. At least five different subfamilies of insulin-like proteins have been identified in vertebrates, represented by insulin (Steiner et al., 1989, in Endocrinology, DeGroot, ed., Philadelphia, Saunders, pp. 1263-1289), insulin-like growth factor (IGF, previously termed somatomedin) (Humbel, 1990, Eur. J. Biochem. 190:445-462), relaxin (Schwabe and Bullesback, 1994, FASEB J. 8:1152-1160), relaxin-like factor (RLF, previously called Leydig cell-specific insulin-like peptide) (Adham et al., 1993, J. Biol. Chem. 268:26668-72; Ivell, 1997, Reviews of Reproduction 2:133-138), and placentin (also known as early placenta insulin-like peptide, or ELIP) (Chassin et al., 1995, Genomics 29:465-470).
Insulin superfamily members in invertebrates have been less extensively analyzed than in vertebrates, but a number of different subgroups have been defined. Such subgroups include molluscan insulin-related peptides (MIP-I to MIP-VII) (Smit et al., 1988, Nature 331:535-538; Smit et al., 1995, Neuroscience 70:589-596), the bombyxins of lepidoptera (originally referred to as prothoracicotropic hormone or PTTH) (Kondo et al., 1996, J Mol. Biol. 259:926-937), and the locust insulin-related peptide (LIRP) (Lagueux et al., 1990, Eur. J. Biochem. 187:249-254). Most recently, there have been descriptions of an exceptionally large insulin-like gene family in the nematode C. elegans (U.S. patent application Ser. No. 09/062,580, filed Apr. 17, 1998 (Attorney Docket No. 7326-059) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Homburger et al; U.S. patent application Ser. No. 09/074,984, filed May 8, 1998 (Attorney Docket No. 7326-068) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Buchman et al; U.S. patent application Ser. No. 09/084,303, filed May 26, 1998 (Attorney Docket No. 7326-069) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Ferguson et al; Duret, et al., 1998, Genome Res. 8:348-353; Brousseau, et al., 1998, Early 1998 East Coast Worm Meeting, abstract 20; Kawano, et al., 1998, Worm Breeder""s Gazette 15(2):47; Pierce and Ruvkun, 1998, Early 1998 East Coast Worm Meeting, abstract 150; Wisotzkey and Liu, 1998, Early 1998 East Coast Worm Meeting, abstract 206). Also, putative orthologs of both vertebrate insulin and IGF have been identified in a tunicate (McRory and Sherwood, 1997, DNA and Cell Biology 116:939-949). Tunicates are thought to be the closest living invertebrate relative to the progenitor from which vertebrates evolved (McRory and Sherwood, 1997, DNA and Cell Biology 16:939-949).
Comparison of the primary sequence of insulin superfamily peptides, cDNAs, and genes, as well as the overall conservation of functional and structural domains of insulin-like genes and proteins, lead to the conclusion that existing members of the insulin superfamily evolved from a common ancestral gene (Blundell and Humbel, 1980, Nature 287:781-787; LeRoith, et al., 1986, Recent Prog. Horm. Res. 42:549-87; Murray-Rust, et al., 1992, BioEssays 14:325-331; LeRoith, et al., 1993, Mol. Reprod. Dev. 35(4):332-8). From the extensive sequence divergence evident among known subfamilies of insulin-like proteins, it is assumed that this is an ancient family of regulatory hormones that evolved to control growth, reproduction and metabolism in early metazoans. However, the precise evolutionary origins of this important family remain unclear.
2.1.1. COMMON STRUCTURAL THEMES
There are common structural themes that unite the insulin superfamily of proteins. Insulin-like peptide hormones are synthesized in vivo as precursor proteins having structures that are variations of the structure schematically represented in FIG. 1. Most precursor forms of the insulin superfamily can be divided into four domains, termed Pre, B, C, and A domains, extending in order from the N-terminus to the C-terminus of a precursor polypeptide (see FIG. 1). Precursors of the IGF subfamily are distinguished by having two additional domains at the C-terminal end, termed D and E domains. The precursors of the locust LIRP protein and some C. elegans insulin-like proteins are distinctive in that they possess another domain, here designated as the F domain, positioned between the Pre domain and the B peptide. The N-terminal Pre domain typically contains a hydrophobic signal sequence which directs secretion of the hormone from cells and is removed by the enzymatic action of a signal peptidase during transit into the endoplasmic reticulum (see the asterisk in FIG. 1). Upon folding, the prohormone undergoes additional processing which, in most cases, involves proteolytic cleavage at two sites that excise the C peptide from the mature hormone (see the two middle arrows illustrated in FIG. 1). These processing steps are mediated by prohormone convertases that cleave at specific positions next to basic residues in the C peptide sequence. As a result, most forms of mature insulin superfamily hormones consist of two polypeptide chains, the A and B peptides, which are covalently joined by disulfide linkages (Sxe2x80x94S) between Cys residues (see Sxe2x80x94S linkages illustrated in FIG. 1). The precise arrangement of Cys residues and disulfide linkages, both between the A and B peptides and within the A peptide, is highly characteristic of the insulin superfamily of hormones. The vast majority of known insulin superfamily members contain six precisely-positioned Cys residues, two in the B chain and four in the A chain, which participate in the formation of three disulfide bonds. Two of these disulfide linkages covalently join the B and A chains (i.e., they form inter-chain bonds), whereas the third disulfide linkage occurs within the A peptide (i.e., as an intra-chain bond) and appears to stabilize a bend in the A chain fold.
The IGF subfamily of hormones has a unique processing pathway. In this subfamily, the connecting C peptide is not removed by processing of the prohormone. Instead, a single proteolytic cleavage event removes the C-terminal E domain (see the right-hand arrow illustrated in FIG. 1). Consequently, mature hormones of the IGF subfamily contain a single polypeptide chain with contiguous B, C, A, and D domains. Despite this difference in proteolytic processing, the disulfide bonding pattern between Cys residues in the IGF subfamily is identical to that of other superfamily members.
In summary, FIG. 1 illustrates the structural organization of precursor forms of the insulin superfamily of hormones. The different domains that make up precursor forms of insulin-like hormones are represented as boxes labeled Pre, F, B, C, A, D, and E, extending from the N-terminus (left) to the C-terminus (right) of the nascent polypeptide chain, respectively. Domains that may remain in a mature hormone are represented as unshaded boxes (the B, A, and D peptide domains) or as lightly hatched (the C or xe2x80x9cconnectingxe2x80x9d peptide domain). By contrast, domains that are removed during proteolytic processing are represented as shaded (the Pre peptide domain and F domain) or as hatched (the E peptide domain). IGF hormones are unique in having D and E peptide domains; these domains are represented as smaller boxes in FIG. 1. Some C. elegans insulin-like proteins are thus far unique in apparently lacking any C peptide sequences and may be produced as a single polypeptide chain consisting of contiguous B and A domains (U.S. patent application Ser. No. 09/062,580, filed Apr. 17, 1998 (Attorney Docket No. 7326-059) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Homburger et al; U.S. patent application Ser. No. 09/074,984, filed May 8, 1998 (Attorney Docket No. 7326-068) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Buchman et al; U.S. patent application Ser. No. 09/084,303, filed May 26, 1998 (Attorney Docket No. 7326-069) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Ferguson et al.; Brousseau, et al., 1998, Early 1998 East Coast Worm Meeting, abstract 20; Duret, et al., 1998, Genome Res. 8(4):348-53; Kawano, et al., 1998, Worm Breeder""s Gazette 15(2):47; Wisotzkey and Liu, 1998, Early 1998 East Coast Worm Meeting, abstract 206). Cleavage sites utilized by proteases during proteolytic processing (i.e., protein maturation) are indicated below the boxes. The asterisk marks the position of cleavage by signal peptidase. Arrows indicate cleavage sites by prohormone convertases. Disulfide bonds (Sxe2x80x94S) are represented above the boxes with lines indicating connections between covalently-bonded Cys residues.
Since the A and B peptide domains constitute common structural segments among all mature insulin superfamily hormones, it is not surprising that these domains are the most highly conserved at the primary sequence level. Even among closely-related members of this superfamily, the domains removed by proteolytic processing (i.e., Pre, C, and E domains) can differ extensively in amino acid sequence composition (McRory and Sherwood, 1997, DNA and Cell Biology 16:939-949; Murray-Rust et al., 1992, BioEssays 14:325-331), in marked contrast to the A and B peptides. Much of the amino acid sequence conservation within the A and B peptide domains reflects residues that play key roles in forming the secondary and tertiary structural elements that are characteristic of the insulin superfamily fold. Aligned sequences of A and B peptide domains from diverse insulin superfamily members are depicted in FIG. 2. This alignment serves to highlight the arrangement of conserved amino acid positions and their relationship to the overall folding pattern of the protein. The three dimensional structures of a number of different insulin superfamily proteins have been determined. Such superfamily proteins include insulin (Hua et al., 1991, Nature 354:238-241), relaxin (Eigenbrot et al., 1991, J. Mol. Biol. 221:15-21), IGF (Cooke et al., 1991, Biochemistry 30:5484-5491), and bombyxin (Nagata et al., 1995, J. Mol. Biol. 253:749-758). The detailed geometry of amino acid side chains in these structures, as well as common secondary and tertiary structural themes, have provided valuable clues about the forces that promote the formation of the characteristic insulin fold. Common features of the main chain fold of insulin-like structures consist of the following: (1) two helices within the A chain joined by a loop; (2) an extended, N-terminal coil within the B chain followed by a tight turn and a central helix; (3) a hydrophobic cluster or xe2x80x9ccorexe2x80x9d that forms an interface between juxtaposed surfaces of the A and B chains; and (4) three disulfide bonds. The common helical regions found in the A and B chains are illustrated in FIG. 2 above the alignment (see xe2x80x9c less than  - - -  greater than xe2x80x9d symbols in FIG. 2).
Beyond the above-described general features of insulin-like structures, there are an number of specific features that are unique to the various subfamilies of insulin-like proteins. Notably, in insulin and IGFs, the main chain following the B peptide central helix forms a tight turn and an extended beta-strand. By contrast, the B chain in both relaxin and bombyxin adopts a fold comprising an extended central helix followed by a coil.
2.1.2. NUMBER AND SPACING OF CYS RESIDUES
The stereotypical arrangement of Cys residues which participate in disulfide linkages within the A and B chains was noted above. It is striking that the exact number and spacing of Cys residues is nearly invariant among insulin-like proteins (see positions B7, B19, A6, A7, A11, and A20, with respect to the human insulin sequence in FIG. 2). Among over 170 sequenced members of the insulin superfamily, only a small minority show deviations from the canonical arrangement of Cys residues. Further, when differences in the arrangement do occur, they tend to be relatively minor. For example, in the case of murine relaxin, the last two Cys residues of the A chain are separated by a spacer of 9 amino acids instead of the canonical 8 amino acids (Evans et al., 1993, J. Mol. Endocrinol. 10:15-23). Another interesting variation occurs in the molluscan insulin-like proteins (MIP-I to -VII). MIP-I appears to have two extra Cys residues, one located N-terminal to the conserved Cys residues within the A chain and the other located N-terminal to the conserved Cys residues of the B chain (see FIG. 2) (Smit et al., 1988, Nature 331:535-538). It has been proposed that this extra pair of Cys residues within MIP-I forms an additional disulfide bond between the A and B chains, thus providing further stability to the folded structure of MIP-I (Smit, et al., 1988, Nature 331:535-538). The most striking examples of variations in Cys positioning within this superfamily come from the insulin-like proteins in the nematode C. elegans (U.S. patent application Ser. No. 09/062,580, filed Apr. 17, 1998 (Attorney Docket No. 7326-059) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Homburger et al; U.S. patent application Ser. No. 09/074,984, filed May 8, 1998 (Attorney Docket No. 7326-068) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Buchman et al; U.S. patent application Ser. No. 09/084,303, filed May 26, 1998 (Attorney Docket No. 7326-069) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Ferguson et al.; Brousseau, et al., 1998, Early 1998 East Coast Worm Meeting, abstract 20; Duret, et al., 1998, Genome Res. 8(4):348-53; Kawano, et al., 1998, Worm Breeder""s Gazette 15(2):47; Pierce and Ruvkun, 1998, Early 1998 East Coast Worm Meeting, abstract 150; Wisotzkey and Liu, 1998, Early 1998 East Coast Worm Meeting, abstract 206). This organism appears to produce over 30 different insulin-like proteins, many of which have unusual Cys arrangements. Such unusual arrangements include the presence of an extra pair of Cys residues, the absence of a conserved pair of Cys residues, and/or altered spacing between Cys residues in either the A or B chain regions. The characteristic insulin core that makes up the interface between the A and B chains is composed of a set of side chains whose conserved hydrophobic nature helps stabilize a tight association. The side chains that participate in the core structure correspond to positions A2, A16, A19, B6, B11, B15, and B18 (see FIG. 2). In addition, the A6-A11 and B19-A20 disulfide bonds are enveloped within the core structure. One other highly-conserved residue within the insulin superfamily is that at B8, which is almost always Gly. The unique flexibility of Gly in this position allows the formation of a tight turn between the extended N-terminus of the B chain and the central helix that immediately follows. Gly residues appear to play a similar role in other positions that promote unique structural features of different insulin subfamily folding patterns. For instance, the Gly at position Bin insulin and IGF appears important in allowing the formation of a tight turn between the central helix and the following beta-strand of the B chain, a hallmark of this subfamily of structures (Blundell et al., 1972, Adv. Protein Chem. 26:279-402). Similarly, a Gly at position A10 in relaxins has been shown to be important for the formation of an exceptionally tight turn between the two A chain helices within the folding pattern of this subfamily (Schwabe and Bullesback, 1994, FASEB J. 8:1152-1160).
2.1.3. RECEPTOR-LIGAND RECOGNITION
An intriguing feature of this diverse family of peptide hormones is the nature of receptor-ligand recognition and the structural basis of its specificity. Although no structures have yet been solved for insulin superfamily receptor-ligand complexes, the issue has been explored through mutational analysis and structure-activity studies of a number of insulin superfamily hormones. The collected results of studies of insulin, relaxin and bombyxin have led to the hypothesis that a common surface is employed by these hormones for receptor-ligand interaction, composed of the central portion of the B chain and the A chain N- and C-termini (Hua, et al., 1991, Nature 354:238-241; Blundell, et al., 1972, Advan. Protein Chem. 26:279-402; Murray-Rust et al., 1992, BioEssays 14:325-331; Nagata et al., 1995, J. Mol. Biol. 253:759-770; Bullesbach et al., 1996, Biochemistry 35:9754-9760; Kristensen et al., 1997, J. Biol. Chem. 272:12978-12983; Schaffer, 1994, Eur. J. Biochem. 221:1127-1132). It appears that insulin and relaxin utilize other structural features for receptor recognition beyond these common elements, specifically, the C-terminus of the B chain in insulin and IGF, and the extended A chain N-terminal helix in relaxin (Nagata et al., 1995, J. Mol. Biol. 253:749-758; Bullesbach et al., 1996, Biochemistry 35:9754-9760; Kristensen et al., 1997, Methods in Cell Biology 44:143-159). Clearly, it is the precise nature of specific amino acid side chains within the receptor recognition surface that contribute to the affinity and specificity of receptor binding. In this regard, a comparison of the residues implicated in receptor recognition for insulin versus relaxin is informative since these two hormones associate with distinct receptor molecules with no evidence for cross-recognition (Rawitch et al., 1980, Int. J. Biochem. 11:357-362).
Residues implicated in insulin receptor recognition include GlyA1, IleA2, ValA3, LeuA13, TyrA19 and AsnA21 on the A chain and ValB12, TyrB16, LeuB17, PheB24, PheB25, and TyrB26 on the B chain (see FIG. 2). A striking feature of this constellation of side chains is that they are largely hydrophobic in character, particularly through the B chain central helix and beta-strand. It is significant that, within the IGF-I sequence, most of the same positions are occupied by either identical or closely-related amino acids to those found in insulin (see FIG. 2). This is consistent with the observation that, although insulin and IGF-I preferentially associate with distinct receptor molecules, there is still measurable cross-recognition by the receptors. Such cross-recognition is believed to be of physiological significance in vivo, perhaps permitting crosstalk between signals controlling growth and metabolism (Humbel, 1990, European Journal of Biochemistry 190:445-462).
In relaxin, by marked contrast, two hydrophilic basic residues have been shown to be critical for receptor recognition. These relaxin residues, ArgB9 and ArgB13 (see FIG. 2), protrude one turn apart from the central B helix (Eigenbrot et al., 1991, J. Mol. Biol. 221:15-21). Not surprisingly, this pair of Arg residues at positions B9 and B13 are rather distinctive for the relaxin subfamily hormones within vertebrates. Other residues implicated in human relaxin II-receptor recognition include TyrA(-1), PheA19, ValB12, GinB15 and IleB16 (Bullesbach and Schwabe, 1988, Int. J. Peptide Protein Res. 32:361-367).
In summary, FIG. 2 illustrates conserved structural features of known insulin superfamily members. The aligned sequences of the B and A chain peptide domains are shown for representative insulin superfamily hormones from the following vertebrates and invertebrates: human insulin (Bell et al., 1979, Nature 29:525-527), human IGF-I (Jansen et al., 1983, Nature 306:609-611), human relaxin 1 (Hudson et al., 1983, Nature 301:628-631), RLF from human (Adham al., 1993, J. Biol. Chem. 268:26668-26672), placentin from human (Chassin et al., 1995, Genomics 29:465-470), bombyxin II from silkworm (Nagasawa et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:5840-5843), MIP from freshwater snail (Smit et al., 1988, Nature 331:535-538), and LIRP from locust (Lagaeux et al., 1990, Eur. J. Biochem. 187:249-254). The numbering scheme shown at the bottom of the figure is for residues of the A and B chains relative to residue numbers for human insulin peptide domains. The nearly invariant positions of the six Cys residues that participate in disulfide bonds are boxed. MIP-I is unusual in having two extra Cys residues which are also individually boxed in that sequence. Other conserved amino acid positions that play important roles in promoting the common insulin superfamily fold are highlighted by shading of the following residue positions: B6, B8, B11, B15, B18, A2, A16, and A19. Three helical regions that comprise the common insulin fold are marked above the alignments using a xe2x80x9c less than  - - -  greater than xe2x80x9d symbol.
2.2. HUMAN INSULIN-LIKE PROTEINS AND THERAPEUTIC APPLICATIONS
As noted above, five different subfamilies of insulin-like hormones are now recognized in humans: insulin, IGF, relaxin, RLF, and placentin. Two of these subfamilies (i.e., RLF and placentin) have been discovered relatively recently and their actual biological roles and corresponding clinical applications remain to be determined. The other three subfamilies (i.e., insulin, IGF, and relaxin) have been studied much more extensively and their roles in regulating growth, differentiation, and metabolism has yielded clinical applications of profound and well-known importance, as described briefly below.
2.2.1. INSULIN
Insulin is the central hormone governing metabolism in vertebrates (reviewed in Steiner et al., 1989, In Endocrinology, DeGroot, eds. Philadelphia, Saunders: 1263-1289). In humans, insulin is secreted by the beta cells of the pancreas in response to elevated blood glucose levels which normally occur following a meal. The immediate effect of insulin secretion is to induce the uptake of glucose by muscle, adipose tissue, and the liver. A longer term effect of insulin is to increase the activity of enzymes that synthesize glycogen in the liver and triglycerides in adipose tissue. Insulin can exert other actions beyond these xe2x80x9cclassicxe2x80x9d metabolic activities, including increasing potassium transport in muscle, promoting cellular differentiation of adipocytes, increasing renal retention of sodium, and promoting production of androgens by the ovary. Defects in the secretion and/or response to insulin are responsible for the disease diabetes mellitus, which is of enormous economic significance. Within the United States, diabetes mellitus is the fourth most common reason for physician visits by patients; it is the leading cause of end-stage renal disease, non-traumatic limb amputations, and blindness in individuals of working age (Warram et al., 1995, In Joslin""s Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea and Febiger, pp. 201-215; Kahn et al., 1996, Annu. Rev. Med. 47:509-531; Kahn, 1998, Cell 92:593-596). Two basic forms of diabetes mellitus occur in humans: type I or insulin-dependent diabetes, and type II or non-insulin-dependent diabetes. A critical problem in managing diabetic patients comes from the phenomenon of insulin resistance, as well as the compounding long term effects of abnormal insulin levels in these individuals. Beyond its role in diabetes mellitus, the phenomenon of insulin resistance has been linked to other pathogenic disorders including obesity, ovarian hyperandrogenism, and hypertension.
The physiologic effects of insulin are mediated by specific association of the peptide hormone with a cell surface receptor, the insulin receptor (IR), with concomitant activation of a signal transduction pathway in responding tissues. The IR has been well-characterized at the molecular level; it is a member of a large family of tyrosine kinase receptors (Ullrich et al., 1985, Nature 313:756-761). IR signaling has been shown to involve a number of intracellular participants (White and Kahn, 1994, J. Biol. Chem. 269: 1-4; Kahn et al., 1998, Cell 92:593-596). These participants include the so-called insulin receptor substrate, or IRS-1, which is phosphorylated by an activated insulin receptor kinase. IRS-1 in turn associates with phosphatidyl-inositol-3-kinase (PI3K). A number of other protein kinases and signaling proteins have been implicated in this signal transduction mechanism and presumably participate in a xe2x80x9ckinase cascadexe2x80x9d that leads to the modification and regulation of a host of intracellular enzymes, structural proteins, and transcription factors. Nonetheless, the precise choreography of events involved in insulin signaling remains vague, and a deeper understanding of such events is likely to have application in surmounting the major clinical problem of insulin resistance. In summary, while clinical issues associated with abnormal insulin levels have raised interest in factors regulating the synthesis, secretion and turnover of insulin, many of the underlying regulatory mechanisms remain to be clarified.
2.2.2. IGF
Humans express two forms of the IGF subfamily of insulin-like hormones, termed IGF-I and IGF-II (Humbel, 1990, Eur. J. Biochem. 190:445-462). These proteins have been found to exert powerful mitogenic effects on a variety of cells and tissues, reflecting their normal physiologic role of promoting growth in developing animals. IGF-I is apparently the primary mediator of growth hormone signaling and, as such, is a major mediator of growth of the skeletal system following birth. IGF-II may have a significant role in fetal growth. Detailed studies with IGF-I, in particular, have led to a variety of significant clinical applications in humans which relate to its growth-promoting and mitogenic properties, including treatment of injuries to the central nervous system, peripheral neuropathy, disorders of the gut, osteoporosis, and congestive heart failure, as well as the acceleration of wound-healing (Gluckman and Nikolics, 1988, xe2x80x9cIGF-1 to improve neural outcomexe2x80x9d, U.S. Pat. No. 5,714,460; Ballard and Read, 1997, xe2x80x9cMethod for treating intestinal diseasesxe2x80x9d, U.S. Pat. No. 5,679,771; Clark et al., 1997, Treatment of congestive heart failurexe2x80x9d, U.S. Pat. No. 5,661,122; Lewis et al., 1997, xe2x80x9cPrevention and treatment of peripheral neuropathyxe2x80x9d, U.S. Pat. Nos. 5,420,112, 5,633,228 and 5,648,335; Burk, 1997, xe2x80x9cComposition and method for the treatment of osteoporosis in mammalsxe2x80x9d, U.S. Pat. No. 5,646,116; Antoniades and Lynch, 1993, xe2x80x9cWound healing using IGF-II and TGFxe2x80x9d, U.S. Pat. No. 5,256,644). Since administration of IGF-I has been shown to increase the growth and size of animals, there are possible applications of this hormone in animal husbandry (Humbel, 1990, Eur. J. Biochem. 190:445-462). As mentioned above, IGFs can elicit insulin-like effects in muscle and adipose tissue, and there is evidence that IGF-I administration may be useful when administered together with insulin in the treatment of diabetes (MacCuish, 1997, xe2x80x9cTreatment of insulin-resistant diabetesxe2x80x9d, U.S. Pat. No. 5,674,845).
2.2.3. RELAXIN
The peptide hormone relaxin was first identified as an active substance in extracts of corpora lutea that induced the separation and relaxation of the pubic symphysis in guinea pigs (Schwabe and Bullesback, 1994, FASEB J. 8:1152-1160). Thus, it was originally believed that the primary physiologic role of relaxin was one associated with promoting parturition during pregnancy. Subsequent studies have confirmed this role in pregnancy for rodents and ruminants. However, the importance of relaxin to the physiology of normal pregnancy in humans is still somewhat unclear (Bani, 1997, Gen. Pharmacol. 28:13-22). Recent studies of relaxin have revealed a more complicated and interesting picture of the spectrum of activities of this peptide hormone. Specifically, relaxin has been found to control growth and differentiation of breast cancer cells in vitro, promote blood vessel dilation, have a chronotropic action on the heart, inhibit histamine release by mast cells, affect pituitary hormone secretion, and regulate fluid balance.
Given this array of physiologic effects, it is not surprising that a number of clinical applications of relaxin have been pursued. These therapeutic applications of relaxin in humans have included the treatment of intractable pain caused by the swelling or dislocation of tissues, as well as the treatment of congestive heart failure, bradycardia, and neurodegenerative diseases (Cronin et al., 1992, xe2x80x9cUse of relaxin in cardiovascular therapyxe2x80x9d, U.S. Pat. No. 5,166,191; Cronin et al., 1995, xe2x80x9cUse of relaxin in the treatment of bradycardiaxe2x80x9d, U.S. Pat. No. 5,478,807; Yue, 1998, xe2x80x9cMethod of treating fibromyalgia with relaxinxe2x80x9d, U.S. Pat. No. 5,707,642). Two forms of relaxin, which are encoded by separate genes, have been identified in humans (Hudson et al., 1983, EMBO J. 3:2333-2339). In contrast to insulin and the IGFs, the specific receptor protein(s) for the relaxins have yet to be characterized at either the DNA or protein sequence level.
2.3. INVERTEBRATE INSULIN-LIKE PROTEINS
Studies of insulin-like molecules in invertebrates have been motivated by the desire to identify proteins which play analogous roles to the well-characterized activities of insulin and IGF in mammals. The first invertebrate insulin-like proteins to be discovered and characterized at the molecular level were the bombyxins of lepidoptera, and they remain the best characterized (Nagasawa et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:5840-5843). Bombyxin, as the name implies, was first identified in extracts of adult heads of the silkworm Bombyx mori. Curiously, it was found that bombyxin stimulated prothoracic glands of the heterologous moth Samia cynthia ricini to synthesize and secrete ecdysteroid hormone. However, no prothoracicotropic activity was observed when bombyxin was injected into Bombyx mori, raising questions about its normal function in this organism (Kiriishi et al., 1992, Zool . Sci. 9:149-155). Bombyxin is produced by neurosecretory cells within the brain of the silkworm and released into the hemolymph. Recent studies with synthetic bombyxin have suggested a role in regulating carbohydrate metabolism with some similarities to the function of insulin in mammals. When injected into neck-ligated larvae, bombyxin reduced the concentration of the major hemolymph sugar, trehalose, and caused elevated activity of trehalase in the midgut and muscle (Satake et al., 1997, Comp. Biochem. Physiol. 188B:349-357). Additional studies have revealed a remarkable array of bombyxin genes. Over 30 separate bombyxin genes have now been identified in the haploid genome of the silkworm (Kondo et al., 1996, J. Mol. Biol. 259:926-937). The bombyxin genes are organized in clusters, and sequence comparisons have led to the categorization of six different gene subtypes. Thus far, all of the bombyxin genes appear to be specifically expressed within four pairs of medial neurosecretory cells in the brain of the silkworm.
DNA-based approaches have been used to isolate insulin-like genes from other invertebrate species, including the LIRP gene from the locust and the MIP-I through MIP-VII series of genes from the freshwater snail (Smit et al., 1998, Prog. Neurobiol. 54:35-54). The biological function of these other invertebrate superfamily members remains largely uncharacterized.
One common theme is that the major site of expression of locust and snail invertebrate insulin-like hormones is in the central nervous system, particularly neurosecretory cells, as has also been observed for the bombyxins of lepidoptera. In the freshwater snail, the cerebral light-green cells, which are the main cells that express the MIP proteins, have been associated with endocrine functions that control glycogen metabolism and the regulation of growth of soft body parts and the shell (Smit et al., 1988, Nature 331:535-538).
2.4. INSULIN SIGNALING IN INVERTEBRATE GENETIC MODEL ORGANISMS
Important issues raised in the preceding discussion regarding the biological function, regulation, and signaling mechanisms of insulin superfamily hormones could best be addressed if these pathways could be analyzed using model genetic organisms. In particular, the facile genetic tools currently available in two model organisms, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, have proven to be of enormous utility in defining the biological function of genes through mutational analysis, as well as for identifying the components of biochemical pathways conserved during evolution with large-scale, systematic genetic screens (Scangos, 1997, Nature Biotechnol. 15:1220-1221; Miklos and Rubin, 1996, Cell 86:521-529). Key discoveries regarding constituents of a number of important human disease pathways, such as the Ras pathway and the pathway controlling programmed cell death, first came from genetic analysis of pathways known to have an evolutionary relation in Drosophila and C. elegans, and later shown to have direct relevance to human biology (Yuan et al., 1993, Cell 75:641-652; Therrien et al., 1995, Cell 83:879-888; Karim et al., 1996, Genetics 143:315-329; Komfeld et al., 1995, Cell 83:903-913; Rubin et al., 1997, xe2x80x9cProtein kinase required for Ras signal transductionxe2x80x9d, U.S. Pat. No. 5,700,675; Steller et al., 1997, xe2x80x9cCell death genes of Drosophila melanogaster and vertebrate homologsxe2x80x9d, U.S. Pat. No. 5,593,879).
2.4.1. THE DAUER STAGE OF C. ELEGANS AND INSULIN SIGNALING
Recent discoveries from studies of C. elegans have led to the identification of components involved in a presumptive insulin signaling pathway. Intriguingly, in C. elegans there are clear connections of this pathway to important aspects of metabolic regulation. This realization has emerged from genetic dissection of the process of dauer larvae formation in the nematode (reviewed in Riddle and Albert, 1997, xe2x80x9cGenetic and environmental regulation of dauer larva developmentxe2x80x9d, In C. elegans II, Riddle et al., eds., Cold Spring Harbor Press, Plainview, N.Y., pp. 739-768), as described further below.
The dauer stage is an alternative developmental stage that is induced when environmental factors are not adequate to promote successful reproduction in C. elegans. There are a number of behavioral, morphologic and metabolic changes that characterize the dauer stage which promote survival of the organism under unfavorable conditions. For example, dauer larvae remain relatively motionless, stop feeding, remain small in size and are reproductively immature. Further, there is increased deposition of fat, a reduction of TCA cycle flux, increased phosphofructokinase activity and increased flux through the glyoxylate cycle in dauer larvae, indicating increased reliance on glycogen and lipid stores as energy reserves in the dauer state (O""Riordan and Burnell, 1989, Comp. Biochem. Physiol. 92B:233-238; O""Riordan and Burnell, 1990, Comp. Biochem. Physiol. 95B:125-130; Wadsworth and Riddle, 1989, Devel. Biol. 132:167-173). Dauer larvae are relatively resistant to detergent, high temperature and oxygen deprivation as compared to normal adults. Remarkably, dauer larvae can live more than four times as long as the normal life span of C. elegans. 
The main environmental cues that control entry into the dauer state are pheromone, food, and temperature. High levels of pheromone (indicative of high population density), low levels of food, and high temperature all favor entry into the dauer stage; reversal of these conditions can induce exit from the dauer stage with resumption of normal organismal development. Clearly, the decision to enter either the dauer pathway or pursue normal development is a major milestone in the life cycle of C. elegans. As such, it likely involves a complex and precise integration of many different physiologic signals. Laser microsurgery has been used to investigate the role of specific cells and tissues in regulating entry into the dauer state (Bargmann and Horvitz, 1991, Science 251:1243-1246).
These cell-killing experiments point to a prominent role for amphid neurons which comprise a pair of chemosensory organs on either side of the head. Killing of specific neurons in the amphid causes a dauer constitutive phenotype, implying that the amphids are responsible for producing a dauer-inhibiting neuronal signal during normal development.
The connection between dauer formation in the nematode and insulin signaling has come from the molecular characterization of the daf-2 gene of C. elegans (Kimura et al., 1997, Science 277:942-946). A daf-2 mutant animal exhibits a dauer constitutive phenotype, and molecular cloning of this gene has revealed that it is a nematode homolog of vertebrate insulin receptors. The physiologic analogy with insulin signaling in vertebrates is that activation of the daf-2 receptor in the nematode corresponds to a xe2x80x9cfedxe2x80x9d state, with the activated daf-2 receptor generating a dauer-inhibiting signal that promotes normal development. Conversely, lack of daf-2 receptor activity corresponds to a xe2x80x9cstarvedxe2x80x9d state, with the lack of inhibitory signal in this pathway favoring entry into the dauer stage. Indeed, studies of other components in the daf-2 signaling pathway have revealed further similarities with insulin signaling in humans. Four other genes, age-1, daf-16, akt-A, and akt-B, have been placed in the same pathway as daf-2 based on analysis of genetic interactions (Morris et al., 1996, Nature 382:536-539; Ogg et al., 1997, Nature 389:994-999; Lin et al., 1997, Science 278:1319-1322). The age-1 gene encodes a nematode homolog of PI3K, and the action of age-1 is required for the propagation of a daf-2 signal, in keeping with the role of PI3K in insulin signaling. Conversely, genetic analysis has shown that the normal role of daf-16 is one of blocking a signal generated by activated daf-2, and daf-i6 has been found to encode a homolog of the HNF-3/forkhead family of transcription factors. In this respect, it is relevant that, in humans, there is the suggestion that insulin mediates some of its effects in target cells by blocking the action of HNF-3 (O""Brien et al., 1995, Mol. Cell. Biol. 15:1747-1758). The akt-A and akt-B genes are thought to provide partially redundant functions within the daf-2 pathway based on preliminary results, and these proteins exhibit homology to protein kinases linked to insulin signaling in vertebrates (Paradis, 1998, Early 1998 East Coast Worm Meeting, abstract 143).
There have been several recent reports describing the identification of insulin-like genes in C. elegans (U.S. patent application Ser. No. 09/062,580, filed Apr. 17, 1998 (Attorney Docket No. 7326-059) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Homburger et al.; U.S. patent application Ser. No. 09/074,984, filed May 8, 1998 (Attorney Docket No. 7326-068) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Buchman et al.; U.S. patent application Ser. No. 09/084,303, filed May 26, 1998 (Attorney Docket No. 7326-069) entitled xe2x80x9cNUCLEIC ACIDS AND PROTEINS OF C. ELEGANS INSULIN-LIKE GENES AND USES THEREOFxe2x80x9d by Ferguson et al.; Brousseau, et al., 1998, Early 1998 East Coast Worm Meeting, abstract 20; Duret, et al., 1998, Genome Res. 8(4):348-53; Kawano, et al., 1998, Worm Breeder""s Gazette 15(2):47; Pierce and Ruvkun, 1998, Early 1998 East Coast Worm Meeting, abstract 150; Wisotzkey and Liu, 1998, Early 1998 East Coast Worm Meeting, abstract 206). These results are striking because of the size and diversity of this subfamily of genes; there are at least 33 insulin-like genes in the C. elegans haploid genome, and many encode proteins with significant and novel deviations from the canonical structure of the insulin superfamily. Specifically, only one known C. elegans insulin-like gene encodes a protein with a clear, excisable C peptide. Further, most of the C. elegans insulin-like proteins have deviations in Cys number or spacing from that found in vertebrate insulin superfamily proteins. At present, it is not certain which of the C. elegans insulin-like proteins are the actual ligand(s) for the daf-2 receptor.
There is another intriguing aspect to the phenotype of nematodes defective in components of the daf-2 pathway with respect to effects on the life-span of the organism (normally about 14 days). Mutations in daf-2 and age-1 can more than double the life-span of animals, even under conditions that do not induce the formation of dauer larvae, and the extension of life-span caused by daf-2 or age-i mutations requires the activity of the daf-16 gene (Lin et al., 1997, Science 278:1319-1322; Tissenbaum and Ruvkun, 1998, Genetics 148:703-717; Larsen et al., 1995, Genetics 139:1567-1583). These findings raise the interesting possibility that detailed genetic analysis of the insulin signaling pathway could reveal new therapeutic approaches with application to aging and longevity in humans.
2.4.2. INSULIN SIGNALING IN DROSOPHILA MELANOGASTER
Early attempts to propagate Drosophila cells in culture revealed a growth factor requirement in defined medium which could be provided by purified bovine insulin, implying the existence of a related endogenous factor in Drosophila (Seecof and Dewhurst, 1974, Cell Differ. 3(1):63-70; Meneses and De Los Angeles Ortiz, 1975, Comp. Biochem. Physiol. A 51(2):483-5; Mosna and Barigozzi, 1976, Experientia 32(7):855-6; Davis and Shearn, 1977, Science 196(4288):438-40; Petersen, et al., 1977, In Vitro 13(1):36-40; Mosna, 1981, Experientia 37(5):466-7; Wyss, 1982, Exp. Cell Res. 139(2):297-307). A bovine and human insulin were found to stimulate the differentiation of Drosophila cells grown in culture (Seecof and Dewhurst, 1974, Cell Differ. 3(1):63-70; Pimentel, et al., 1996, Biochem. Biophys. Res. Commun. 226(3):855-61). One report described the presence of an xe2x80x9cinsulin-like activityxe2x80x9d in unpurified Drosophila extracts that elicited a hypoglycemic effect when injected into mice, although the activity was not particularly strong (Meneses and De Los Angeles Ortiz, 1975, Comp. Biochem. Physiol. A. 51(2):483-5). Another group (LeRoith, et al., 1981, Diabetes 30(1):70-6) fractionated an insulin-like material from Drosophila based on immunoreactivity and showed that this material had insulin-like activity on isolated rat adipocytes. Also, polyclonal antibodies raised against bovine/porcine insulin were used to localize insulin-immunoreactive material in Drosophila tissue (Gorczyca, et al., 1993, J. Neurosci. 13(9):3692-704), and specific insulin-inmunoreactive substances were detected at neuromuscular junctions and in the central nervous system. However, these substances were not characterized further to validate that they correspond to bonafide insulin proteins at the level of primary protein sequence. Indeed, despite this long history of phenomenological evidence for insulin-like activities, true insulin-like genes and proteins in Drosophila have not been identified and characterized at the sequence level.
More compelling evidence for evolutionary conservation of insulin-like signaling pathways in Drosophila has come from the identification of an apparent homolog of the insulin receptor (Petruzzelli et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:4710-4714). One insulin receptor homolog has been characterized thus far in Drosophila, termed InR (insulin receptor) also known as DIR (Drosophila insulin receptor) (Ruan et al., 1995, J. Biol. Chem. 270:4236-4243), which exhibits extensive homology with vertebrate insulin and IGF receptors in both the extracellular ligand-binding domain and the intracellular tyrosine kinase domain. InR is larger than the human insulin receptor protein due to extensions at both the N- and C-termini of the polypeptide chain. It is interesting that the additional C-terminal segment of the InR shares sequence features with IRS-1, one of the substrates of the insulin receptor tyrosine kinase in mammals. Genetic analysis of InR function in Drosophila has revealed that it is an essential gene with an apparent role in the development of the epidermis and nervous system, as well as growth control (Fernandez et al., 1995, EMBO J. 14:3373-3384). Flies that are homozygous for mutations in InR generally exhibit an embryonic lethal phenotype, but flies bearing certain heteroallelic combinations of InR mutations live to adulthood and the surviving animals have about 50% the normal body weight (Garafalo, Chen, et al., 1996, Endocrinology 137(3):846-56). This result is reminiscent of a similar phenotype observed in mutant mice lacking functional IGF-I receptor genes (Liu, et al., 1993, Cell 75(1):59-72). Aside from this potential role of InR in growth regulation, the role, if any, that InR may have in metabolic regulation in Drosophila remains unclear. The ligand binding specificity of InR has been examined using in vitro assays for receptor activation/phosphorylation, and competitive binding of test ligands compared to porcine insulin (Fernandez-Almonacid and Rosen, 1987, Mol. Cell Biol. 7(8):2718-27). Curiously, the results of this study indicated that InR binds vertebrate insulin, and does not apparently recognize vertebrate IGF-I or IGF-II, or even bombyxin-II from the silkworm, implying that the natural Drosophila ligands for InR may bear more structural resemblance to vertebrate insulin than to these other insulin superfamily proteins.
Two other Drosophila genes have been tentatively placed downstream of InR in signaling for growth control, based on preliminary data. Dominant negative and constitutively active forms of Drosophila Pi3K92E, encoding PI3-kinase cause growth defects when expressed in the fly eye and wing that are consistent with action downstream of InR (Leevers et al., 1996, EMBO J. 15(23):6584-94) and have been reported to interact genetically with InR mutants (Leevers et al., 1998, A. Conf. Dros. Res. 39:31). In addition, the Drosophila chico gene encodes a homologue of IRS-1. Mutations in chico are semi-lethal, with surviving adults having small body size consistent with the data on InR mutants (abstract Riesgo-Escovar, et al., 1998, A. Conf. Dros. Res. 39:32).
Recently, a Drosophila insulin-like gene has been isolated and characterized (see U.S. patent application Ser. No. 09/201,226 (Attorey Docket No. 7326-077 filed evendate herewith now U.S. Pat. No. 6,135,942 issued Oct. 24, 2000, entitled xe2x80x9cNUCLCEIC ACIDS AND PROTEINS OF A D. MELANOGASTER INSULIN-LIKE GENE AND USES THEREOFxe2x80x9d by Maria Leptin, which is incorporated herein by reference in its entirety).
2.4.3. UNANSWERED QUESTIONS
The structural homologies of components of the Drosophila InR pathway with those involved in insulin signaling in mammals, as well as the function of the InR pathway in controlling growth, and the circumstantial evidence for Drosophila insulin-like activities, raise critical questions with respect to further analysis of this pathway and its potential applications. For example, are there, in fact, insulin superfamily hormones in Drosophila? If so, how diverse is the insulin superfamily in Drosophila in terms of structure and function? Particularly, are Drosophila insulin-like proteins closer in structure and function to their vertebrate counterparts than those found in the nematode C. elegans? Further, what specific Drosophila insulin-like protein(s) interact with the InR receptor, or otherwise affect growth control? Are there other receptors for Drosophila insulin-like proteins aside from InR that are involved in regulating other functions, such as metabolism, development, reproduction, or longevity? Finally, how are the synthesis, activity and turnover of insulin-like proteins regulated in Drosophila? Answers to the foregoing questions would be much desired.
The present invention relates to the nucleotide sequences of D. melanogaster insulin-like genes, the amino acid sequences of their encoded proteins, and derivatives (e.g., fragments) and analogs thereof. Nucleic acids capable of hybridizing to or complementary to the foregoing nucleotide sequences are also provided. The invention also relates to a method of identifying genes that are modified by, or that participate in signal transduction with, D. melangaster insulin-like genes. The invention also relates to derivatives and analogs of D. melangaster insulin-like genes which are functionally active, i.e., which are capable of displaying one or more known functional activities associated with a full-length (wild-type) insulin-like protein. Such functional activities include but are not limited to antigenicity (ability to bind, or to compete for binding, to an anti-insulin antibody), immunogenicity (ability to generate antibody which binds to insulin), and ability to bind (or compete for binding) to a receptor for insulin (e.g., that is encoded by the D. melanogaster insulin receptor-like gene InR). The invention further relates to fragments (and derivatives and analogs thereof) of an insulin-like protein which comprise one or more domains of an insulin-like protein. Antibodies to an insulin-like protein, derivatives and analogs of an insulin-like protein, are additionally provided. Methods of production of the insulin-like proteins, derivatives and analogs, e.g., by recombinant means, are also provided. Methods to identify the biological function of a Drosophila insulin-like gene are provided, including various methods for the functional modification (e.g., overexpression, underexpression, mutation, knock-out) of one gene, or of two or more genes simultaneously. Methods to identify a Drosophila gene which modifies the function of, and/or functions in a downstream pathway from, an insulin-like gene are provided. The invention further provides for use of Drosophila insulin-like proteins as a media additive or pesticide.
This invention provides a purified protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2). The invention further provides a purified protein comprising amino acid sequence numbers 30-120 as depicted in FIG. 5 (SEQ ID NO:2).
This invention provides a purified protein comprising an amino acid sequence as depicted in FIG. 6 (SEQ ID NO:4). The invention further provides a purified protein comprising amino acid sequence numbers 30-154 as depicted in FIG. 6 (SEQ ID NO:4).
This invention provides a purified protein comprising an amino acid sequence as depicted in FIG. 7 (SEQ ID NO:6). The invention further provides a purified protein comprising amino acid sequence numbers 27-137 as depicted in FIG. 7 (SEQ ID NO:6).
This invention provides a purified protein, the amino acid sequence of which consists of amino acids numbers 1-120 as depicted in FIG. 5 (SEQ ID NO:2). This invention further provides a purified protein, the amino acid sequence of which consists of amino acids numbers 30-120 as depicted in FIG. 5 (SEQ ID NO:2).
This invention provides a purified protein, the amino acid sequence of which consists of amino acids numbers 1-154 depicted in FIG. 6 (SEQ ID NO:4). This invention further provides a purified protein, the amino acid sequence of which consists of amino acids numbers 30-154 as depicted in FIG. 6 (SEQ ID NO:4).
This invention provides a purified protein, the amino acid sequence of which consists of amino acids numbers 1-137 as depicted in FIG. 7 (SEQ ID NO:6). This invention provides a purified protein, the amino acid sequence of which consists of amino acids numbers 27-137 as depicted in FIG. 7 (SEQ ID NO:6).
This invention provides a purified protein consisting of a B peptide domain defined by amino acid sequence numbers 30-54 as depicted in FIG. 5 (SEQ ID NO:2), linked by one or more disulfide bonds to an A peptide domain defined by amino acid sequence numbers 92-120 as depicted in FIG. 5 (SEQ ID NO:2).
This invention provides a purified protein comprising a B peptide domain defined by amino acid sequence numbers 30-54 as depicted in FIG. 5 (SEQ ID NO:2).
This invention provides a purified protein comprising an A peptide domain defined by amino acid sequence numbers 92-120 as depicted in FIG. 5 (SEQ ID NO:2).
This invention provides a purified protein consisting of a B peptide domain defined by amino acid sequence numbers 30-69 as depicted in FIG. 6 (SEQ ID NO:4), linked by one or more disulfide bonds to an A peptide domain defined by amino acid sequence numbers 128-154 as depicted in FIG. 6 (SEQ ID NO:4).
This invention provides a purified protein consisting of a B peptide domain defined by amino acid sequence numbers 30-69 as depicted in FIG. 6 (SEQ ID NO:4), linked by one or more disulfide bonds to an A peptide domain defined by amino acid sequence numbers 129-154 as depicted in FIG. 6 (SEQ ID NO:4).
This invention provides a purified protein comprising a B peptide domain defined by amino acid sequence numbers 30-69 as depicted in FIG. 6 (SEQ ID NO:4).
This invention provides a purified protein comprising an A peptide domain defined by amino acid sequence numbers 128-154 as depicted in FIG. 6 (SEQ ID NO:4).
This invention provides a purified protein comprising an A peptide domain defined by amino acid sequence numbers 129-154 as depicted in FIG. 6 (SEQ ID NO:4).
This invention provides a purified protein consisting of a B peptide domain defined by amino acid sequence numbers 27-50 as depicted in FIG. 7 (SEQ ID NO:6), linked by one or more disulfide bonds to an A peptide domain defined by amino acid sequence numbers 108-137 as depicted in FIG. 7 (SEQ ID NO:6).
This invention provides a purified protein consisting of a B peptide domain defined by amino acid sequence numbers 27-49 as depicted in FIG. 7 (SEQ ID NO:6), linked by one or more disulfide bonds to an A peptide domain defined by amino acid sequence numbers 108-137 as depicted in FIG. 7 (SEQ ID NO:6).
This invention provides a purified protein comprising a B peptide domain defined by amino acid sequence numbers 27-50 as depicted in FIG. 7 (SEQ ID NO:6).
This invention provides a purified protein comprising a B peptide domain defined by amino acid sequence numbers 27-49 as depicted in FIG. 7 (SEQ ID NO:6).
This invention provides a purified protein comprising an A peptide domain defined by amino acid sequence numbers 108-137 as depicted in FIG. 7 (SEQ ID NO:6).
This invention provides a purified fragment comprising at least 10 contiguous amino acids of a protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), which fragment is capable of being bound by an antibody to said protein.
This invention provides a purified first protein comprising at least 10 contiguous amino acids of a second protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), which first protein has only an insertion, deletion, or substitution relative to the sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), and which first protein is capable of being bound by an antibody to said second protein.
This invention provides a purified protein comprising a fragment comprising at least 10 contiguous amino acids of a protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), which fragment is capable of being bound by an antibody to said protein.
This invention provides a purified fragment of a protein consisting of an amino acid sequence depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4) or FIG. 7 (SEQ ID NO:6), said fragment comprising (a) at least 10 contiguous amino acids; and (b) a domain of said protein selected from the group consisting of a B peptide domain and an A peptide domain.
This invention provides a chimeric protein comprising the fragment comprising at least 10 contiguous amino acids of a protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), which fragment is capable of being bound by an antibody to said protein, fused by a covalent bond to at least a portion of a second protein, which second protein is not said protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6(SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6). In one embodiment, the chimeric protein comprising the fragment is fused by a covalent bond to at least a portion of a second protein, which second protein is not an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6(SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6). In another embodiment, the fragment is fused by a covalent bond to at least a portion of a second protein, which second protein is not a D. melangaster insulin-like protein.
This invention provides a purified molecule comprising a fragment of at least contiguous amino acids of a protein defined by an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, which fragment is capable of being bound by an antibody to said protein defined by the sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
This invention provides a chimeric protein comprising a fragment of a protein consisting of an amino acid sequence depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4) or FIG. 7 (SEQ ID NO:6), said fragment comprising (a) at least 10 contiguous amino acids; and (b) a domain of said protein selected from the group consisting of a B peptide domain and an A peptide domain, fused by a covalent bond to at least a portion of a second protein, which second protein is not said protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6(SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6). In another embodiment, the fragment is fused by a covalent bond to at least a portion of a second protein, which second protein is not a D. melangaster insulin-like protein. In yet another embodiment, the fragment is capable of being bound by an antibody to a protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6).
This invention provides a purified molecule comprising a purified fragment of a protein consisting of an amino acid sequence depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4) or FIG. 7 (SEQ ID NO:6), said fragment comprising (a) at least contiguous amino acids; and (b) a domain of said protein selected from the group consisting of a B peptide domain and an A peptide domain.
This invention provides a purified antibody or derivative thereof, containing an idiotype capable of immunospecific binding to a protein consisting of an amino acid sequence depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4) or FIG. 7 (SEQ ID NO:6) and not to an insulin-like protein of another species. In one embodiment, the antibody is polyclonal. In another embodiment, the antibody is monoclonal.
This invention provides an isolated nucleic acid comprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1) or FIG. 7 (SEQ ID NO:5).
This invention provides an isolated nucleic acid comprising a nucleotide sequence as depicted in FIG. 6 (SEQ ID NO:3), wherein said nucleic acid is less than 15 kilobases.
This invention provides an isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), wherein said nucleic acid is less than 15 kilobases.
This invention provides an isolated RNA molecule comprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), wherein the base U (uracil) is substituted for the base T (thymine) of said sequence.
This invention provides an isolated RNA molecule comprising a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6).
This invention provides an isolated first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid defined by a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), wherein said first nucleic acid is less than 15 kilobases. In one embodiment, the first nucleic acid encodes a first protein capable of being bound by an antibody to a second protein defined by the amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6).
This invention provides an isolated first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid defined by the reverse complement a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), wherein said first nucleic acid is less than 15 kilobases.
This invention provides a purified protein encoded by a first nucleic acid capable of hybridizing under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid defined by the reverse complement of a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), wherein said protein is capable of being bound by an antibody to a second protein defined by an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6, (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6).
This invention provides an isolated first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid defined by a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), wherein said first nucleic acid is less than 15 kilobases. In one embodiment, the nucleic acid encodes a D. melangaster insulin-like protein or a fragment of at least 10 contiguous amino acids of said protein.
This invention provides an isolated first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid defined by the reverse complement of a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), wherein said first nucleic acid is less than 15 kilobases.
This invention provides an isolated nucleic acid comprising a nucleotide sequence that is the reverse complement of a nucleotide sequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6).
This invention provides a method of producing a protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a recombinant nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1) or FIG. 7 (SEQ ID NO:5), such that the protein encoded by said nucleotide sequence is expressed by the cell; and (b) recovering the expressed protein. In one embodiment, the purified protein produced by the method is provided.
This invention provides a method of producing a protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a recombinant nucleotide sequence as depicted in FIG. 6 (SEQ ID NO:3) of less than 15 kilobases, such that the protein encoded by said nucleotide sequence is expressed by the cell; and (b) recovering the expressed protein. In one embodiment, the purified protein produced by the method is provided.
This invention provides a method of producing a protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a recombinant nucleotide sequence of less than 15 kilobases encoding a protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), such that the encoded protein is expressed by the cell; and (b) recovering the expressed protein. In one embodiment, the purified protein produced by the method is provided.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a D. melangaster insulin-like protein comprising identifying an effect of a mutated or abnormally expressed D. melangaster insulin-like gene which encodes a D. melangaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), in a D. melangaster animal. In one embodiment, the effect is determined by an assay selected from the group consisting of a developmental assay, an energy metabolism assay, a growth rate assay, a reproductive capacity assay, a lethality assay, a sterility assay, a brood size assay, a life span assay, a locomotion assay, a body shape assay, a body plan assay, a body size assay, a body weight assay, a cell size assay, a cell division assay, a feeding assay, a developmental rate assay, and a morphogenesis assay. In another embodiment, the gene is mutated or abnormally expressed using a technique selected from the group consisting of radiation mutagenesis, chemical mutagenesis, transposon mutagenesis, antisense and double-stranded RNA interference.
This invention provides a method of identifying a phenotype associated with mutation or abnormal expression of a D. melangaster insulin-like protein comprising: (a) mutating or abnormally expressing a D. melangaster insulin-like gene which encodes a D. melanogaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), in a D. melanogaster animal, wherein the nucleotide sequence comprising SEQ ID NO:3 does not contain genomic sequence naturally contiguous with SEQ ID NO:3 of greater than 15 kilobases; and (b) identifying an effect of the gene mutated or abnormally expressed. In one embodiment, the effect is identified by an assay selected from the group consisting of a developmental assay, an energy metabolism assay, a growth rate assay, a reproductive capacity assay, a lethality assay, a sterility assay, a brood size assay, a life span assay, a locomotion assay, a body shape assay, a body plan assay, a body size assay, a body weight assay, a cell size assay, a cell division assay, a feeding assay, a developmental rate assay, and a morphogenesis assay. In another embodiment, the gene is mutated or abnormally expressed using a technique selected from the group consisting of radiation mutagenesis, chemical mutagenesis, transposon mutagenesis, antisense and double-stranded RNA interference.
This invention provides a recombinant cell containing a recombinant nucleic acid vector of less than 15 kilobases comprising a nucleotide sequence as depicted in FIG. 6 (SEQ ID NO:3).
This invention provides a recombinant cell containing a recombinant nucleic acid vector comprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1) or FIG. 7 (SEQ ID NO:5).
This invention provides a vector comprising (a) a nucleotide sequence as depicted in FIG. 6 (SEQ ID NO:3), and (b) an origin of replication, wherein said vector does not contain genomic sequence naturally contiguous with SEQ ID NO:3 of greater than kilobases. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, and SEQ ID NO:5 and an origin of replication. In one embodiment, the nucleotide sequence is operably linked to a heterologous promoter.
This invention provides a method of identifying a gene-of-interest as capable of modifying a function of a D. melangaster insulin-like gene comprising: (a) constructing a first mutant fly having a first mutation in a D. melangaster insulin-like gene which encodes a D. melangaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6) and a second mutation in the gene-of-interest; and (b) determining whether the phenotype displayed by the first mutant fly is different from the phenotype of a second mutant fly having said first mutation but not said second mutation, in which the displaying of a phenotype by the first mutant fly that is different from said second mutant fly identifies the gene-of-interest as capable of modifying the function of the D. melangaster insulin-like gene. In one embodiment, the first mutant fly is produced using a technique selected from the group consisting of radiation mutagenesis, chemical mutagenesis, transposon mutagenesis, antisense and double-stranded RNA interference. In another embodiment, the phenotype is selected from the group consisting of lethality, sterility, altered brood size, altered life span, altered locomotion, altered body shape, altered body plan, altered body size, altered body weight, altered cell size, altered cell division, altered feeding, altered development, altered metabolism, altered glycogen synthesis, altered glycogen storage, altered glycogen degradation, altered lipid synthesis, altered lipid storage, altered lipid degradation, altered levels of carbohydrate in hemolymph, altered levels of lipid in hemolymph, altered morphogenesis of organs, altered morphogenesis of tissues of the gonad, altered morphogenesis of the nervous system, altered fat body, altered hemocytes, altered morphogenesis of the peripheral sensory organs, altered imaginal discs, altered eye development, altered wing development, altered leg development, altered bristle development, altered antennae development, altered gut development, and altered musculature. In a further embodiment, the altered organ morphogenesis phenotype involves an organ selected from the group consisting of gonad, nervous system, fat body, hemocytes, peripheral sensory organs, imaginal discs, eye, wing, leg, antennae, gut, musculature, and bristle. In yet another embodiment, the fly having the altered phenotype is assayed for activity of a gene affecting body size selected from the group consisting of InR, chico, Pi3K92, Akt1, 14-3-3z, Lar, Pk61C, Glut3, Ide, shaggy, s6k, Ras85D, drk, Sos, rl, and Dsor1. In yet another embodiment, the gene-of-interest is a homolog of an insulin signaling pathway gene from vertebrates. In another embodiment, the gene-of-interest is selected from the group consisting of InR, chico, Pi3K92, Akt1, 14-3-3z, Lar, Pk61C, Glut3, Ide, shaggy, s6k, Ras85D, drk, Sos, rl, and Dsor1.
This invention provides a D. melangaster animal having a first mutation in a D. melangaster insulin-like gene comprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), and a second mutation in a different gene that is a homolog of an insulin signaling pathway gene from vertebrates.
This invention provides a method of studying a function of a D. melanogaster insulin-like gene comprising: (a) mis-expressing a wild-type or mutant D. melanogaster insulin-like gene which encodes a D. melangaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6) in a transgenic fly by driving expression with a homologous or heterologous promoter; and (b) detecting a phenotype in said transgenic fly, so as to study the function of the D. melangaster insulin-like gene. In one embodiment, the heterologous promoter driving mis-expression is selected from the group consisting of a heat shock factor-responsive promoter, a GAL4-responsive promoter, a tTA-responsive promoter, a glass-responsive promoter, an eyeless enhancer-regulated promoter, a dpp enhancer-regulated promoter, and a vestigial enhancer-regulated promoter. In another embodiment, said transgenic fly mis-expressing the D. melangaster insulin-like gene further has a mutation in a gene selected from the group consisting of InR, chico, Pi3K92, Akt1, 14-3-3z, Lar, Pk61C, Glut3, Ide, shaggy, s6k, Ras85D, drk, Sos, rl, and Dsor1. In another embodiment, said transgenic fly mis-expressing the D. melangaster insulin-like gene is assayed for a change in a phenotype selected from the group consisting of lethality, sterility, altered brood size, altered life span, altered locomotion, altered body shape, altered body plan, altered body size, altered body weight, altered cell size, altered cell division, altered feeding, altered development, altered metabolism, altered glycogen synthesis, altered glycogen storage, altered glycogen degradation, altered lipid synthesis, altered lipid storage, altered lipid degradation, altered levels of carbohydrate in hemolymph, altered levels of lipid in hemolymph, altered morphogenesis of organs, altered morphogenesis of tissues of the gonad, altered morphogenesis of the nervous system, altered fat body, altered hemocytes, altered morphogenesis of the peripheral sensory organs, altered imaginal discs, altered eye development, altered wing development, altered leg development, altered bristle development, altered antennae development, altered gut development, and altered musculature.
This invention provides a method of detecting the effect of expression of a D. melangaster insulin-like gene which encodes a D. melangaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), on an insulin signaling pathway comprising: (a) mutating or abnormally expressing a wild-type D. melangaster insulin-like gene that encodes a protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6) in a fly already having a mutation in the insulin signaling pathway that displays a phenotype-of-interest; and (b) detecting the effect of step (a) on the phenotype-of-interest, so as to detect the effect of expression of the D. melangaster insulin-like gene. In one embodiment, the mutation in the insulin signaling pathway is in a gene selected from the group consisting of InR, chico, Pi3K92, Akt1, 14-3-3z, csw, Lar, Pk61C, Glut3, Ide, shaggy, s6k, Ras85D, drk, Sos, rl, an Dsor1.
This invention provides a method of identifying a molecule that binds to a ligand selected from the group consisting of (i) a D. melangaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), (ii) a fragment of the D. melangaster insulin-like protein comprising a domain of the protein, and (iii) a nucleic acid encoding the D. melanogaster insulin-like protein or fragment, the method comprising: (a) contacting the ligand with a plurality of molecules under conditions conducive to binding between the ligand and the molecules; and (b) identifying a molecule within the plurality that binds to the ligand. In one embodiment, the domain of the D. melangaster insulin-like protein is selected from the group consisting from a signal peptide domain, a pre peptide domain, a B peptide domain, a C peptide domain and an A peptide domain.
This invention provides a modified, isolated D. melangaster animal in which a D. melangaster insulin-like gene which encodes a D. melangaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6) which has been deleted or inactivated by recombinant methods, or a progeny thereof containing the deleted or inactivated gene.
This invention provides a modified, isolated D. melangaster animal in which insulin-like gene has been deleted or inactivated by a method selected from the group consisting of radiation mutagenesis, chemical mutagenesis, transposon mutagenesis, antisense and double-stranded RNA interference.
This invention provides a recombinant non-human animal containing a D. melanogaster insulin-like transgene which encodes a D. melangaster insulin-like protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6). In one embodiment, the D. melangaster insulin-like transgene is under the control of a promoter that is not the native promoter of the transgene.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, wherein the protein is characterized as having a cleavable C peptide, and B and A chains, and as having the same number and relative spacing of Cys residues as found in vertebrate insulin-like proteins. In one embodiment, the B and A chain domains of the protein are not proteolytically cleaved into separate chains.
This invention provides a purified protein encoded by a first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), wherein the protein is characterized as having a cleavable C peptide separating the B and A chains. In one embodiment, the B and A chain domains of the protein are not proteolytically cleaved into separate chains.
This invention provides a method of identifying a molecule that alters the expression level of a D. melangaster insulin-like gene corresponding to a cDNA sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), which method comprises: (a) contacting a transgenic fly cell with one or more molecules, said transgenic fly cell having a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA sequence, operably linked to a reporter gene; and (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules. In one embodiment, the reporter gene encodes a protein selected from the group consisting of green fluorescent protein, lacZ protein, cre protein, FLP protein, reaper protein, hid protein, GAL4 protein, and tTA protein.
This invention provides a method of identifying a molecule that binds to a promoter or enhancer of a D. melangaster insulin-like gene corresponding to a cDNA sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), which method comprises: (a) contacting a transgene comprising a promoter or enhancer region of genomic DNA from 1 base to 6 kilobases upstream of the start codon of the cDNA sequence operably linked to a reporter gene, with the molecule; and (b) determining whether the level of expression of the reporter gene is altered relative to the level of expression of the reporter gene in the absence of the one or more molecules. In one embodiment, the reporter gene encodes a protein selected from the group consisting of green fluorescent protein, lacZ protein, cre protein, FLP protein, reaper protein, hid protein, GAL4 protein, and tTA protein.
This invention provides a purified genomic nucleic acid consisting of a nucleotide sequence as depicted in FIG. 4 (SEQ ID NO:7).
This invention further provides a purified genomic nucleic acid consisting of a nucleotide sequence of less than 15 kilobases and comprising nucleotide numbers 1 to 967 as depicted in FIG. 4 (SEQ ID NO:7), or at least 20 contiguous nucleotides of SEQ ID NO:7.
This invention provides a purified genomic nucleic acid consisting of a nucleotide sequence of less than 15 kilobases and comprising nucleotide numbers 1583 to 11120 as depicted in FIG. 4 (SEQ ID NO:7) or at least 20 contiguous nucleotides of SEQ ID NO:7.
This invention provides a cell culture medium or medium supplement comprising (a) a sterile liquid carrier, and (b) a protein or fragment thereof, functional in promoting cell growth, survival, or differentiation, said protein comprising at least 10 contiguous amino acids as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6).
This invention provides a cell culture medium or medium supplement comprising (a) a sterile liquid carrier, and (b) a protein encoded by a first nucleic acid which hybridizes under conditions selected from the group consisting of high stringency, moderate stringency and low stringency, to a second nucleic acid, which second nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, wherein the protein is characterized as having a cleavable C peptide, and B and A chains, and as having the same number and relative spacing of Cys residues as found in vertebrate insulin-like proteins, or a fragment thereof, functional in promoting cell growth, survival, or differentiation comprising at least 10 contiguous amino acids of said A chain or B chain of said protein. In one embodiment, the cell culture medium or medium supplement further comprises growth factors, vitamins, carbohydrates, antibiotics, antimicrobial agents, or salts. In another embodiment, the protein or fragment is purified.
This invention provides a method for growing, maintaining or differentiating a cell in culture comprising contacting the cell with an effective amount of a protein, said protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), or a fragment of said protein functional in promoting cell growth, survival, or differentiation comprising at least 10 contiguous amino acids of an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6). In one embodiment the protein or fragment is purified. In another embodiment, the cell is selected from the group consisting of an animal cell and a plant cell. In still another embodiment, the cell is a D. melangaster cell.
This invention provides a pesticide formulation comprising (a) a carrier, and (b) a protein, said protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6), or a pesticidal fragment of said protein, said fragment comprising at least 10 contiguous amino acids of said protein. In one embodiment, the protein or fragment is purified. In another embodiment, the carrier is selected from the group consisting of water, organic solvent, inorganic solvent, talc, pyrophyllite, synthetic fine silica, attapugus clay, kieselguhr chalk, diatomaceous earth, lime, calcium carbonate, bontonite, fuller""s earth, cottonseed hulls, wheat flour, soybean flour, pumice, tripoli, wood flour, walnut shell flour, redwood flour, and lignin. In another embodiment, this invention provides a method for protecting a plant or animal against a pest comprising contacting the plant or animal with the pesticide formulation.
This invention provides a pesticide formulation comprising (a) a carrier, and (b) a purified nucleic acid encoding a protein comprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:2), FIG. 6 (SEQ ID NO:4), or FIG. 7 (SEQ ID NO:6).
This invention provides a pesticide formulation comprising (a) a carrier, and (b) a nucleic acid, said nucleic acid comprising at least a portion of a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:1), FIG. 6 (SEQ ID NO:3), or FIG. 7 (SEQ ID NO:5), said portion encoding a protein functional as a pesticide. In one embodiment, the nucleic acid is purified. In another embodiment, the carrier is selected from the group consisting of water, organic solvent, inorganic solvent, talc, pyrophyllite, synthetic fine silica, attapugus clay, kieselguhr chalk, diatomaceous earth, lime, calcium carbonate, bontonite, fuller""s earth, cottonseed hulls, wheat flour, soybean flour, pumice, tripoli, wood flour, walnut shell flour, redwood flour, and lignin. In another embodiment, the nucleic acid is a plasmid expression vector. In a further embodiment, the nucleic acid is contained in a recombinant virus. In a further embodiment, the recombinant virus is a baculovirus. In yet another embodiment, this invention provides a method for protecting a plant or animal against a pest comprising contacting the plant or animal with the pesticide formulation.